Serological Studies of Neurologic Helminthic Infections in Rural Areas of Southwest Cameroon: Toxocariasis,Cysticercosis and Paragonimiasis

Background

Both epilepsy and paragonimiasis had been known to be endemic in Southwest Cameroon. A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old) were selected on a voluntary basis. Among 14 people (8.3%) with history of epilepsy, only one suffered from paragonimiasis. Therefore, we challenged to check antibody responses to highly specific diagnostic recombinant antigens for two other helminthic diseases, cysticercosis and toxocariasis, expected to be involved in neurological diseases. Soil-transmitted helminthic infections were also examined.

Methodology/Principal Findings

Fecal samples were collected exclusively from the 168 people. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%), 72 (42.8%), and 19 (11.3%) persons, respectively. Serology revealed that 61 (36.3%), 25 (14.9%) and 2 (1.2%) of 168 persons showed specific antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. By contrast, 20 people without any symptoms as well as additional 20 people from Japan showed no antibody responses. Among the 14 persons with epilepsy, 5 persons were seropositive to the antigen specific to Toxocara, and one of them was simultaneously positive to the antigens of Paragonimus. The fact that 2 children with no history of epilepsy were serologically confirmed to have cysticercosis strongly suggests that serological survey for cysticercosis in children is expected to be useful for early detection of asymptomatic cysticercosis in endemic areas.

Conclusions/Significance

Among persons surveyed, toxocariasis was more common than paragonimiasis, but cysticercosis was very rare. However, the fact that 2 children were serologically confirmed to have cysticercosis was very important, since it strongly suggests that serology for cysticercosis is useful and feasible for detection of asymptomatic cysticercotic children in endemic areas for the early treatment.     

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T_m > 70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners.     

Spindle positioning in mammalian oocytes

To preserve the maternal stores accumulated during oogenesis for further embryo development, oocytes divide asymmetrically which minimizes the volume of cytoplasm lost with each set of haploid genome. To ensure asymmetric division to occur, oocytes have to position their division spindle asymmetrically as well as tailor the size of daughter cells to the chromatin mass. In this review, we will discuss the recent advances in the field, with emphasis on the control mechanisms involved in meiotic spindle positioning in mammalian oocytes.     

Thioredoxin m4 Controls Photosynthetic Alternative Electron Pathways in Arabidopsis

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF.In plant thylakoids, photosynthesis involves a linear electron flow (LEF) from water to NADP⁺ via PSII, cytochrome b₆/f, PSI, and soluble carriers. LEF produces NADPH and generates a transthylakoidal electrochemical proton gradient that drives the synthesis of ATP. Besides LEF, cyclic electron flow (CEF) can also occur, involving only PSI (for review, see Johnson, 2011; Kramer and Evans, 2011). These additional reactions include two main distinct pathways involving either the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (Munekage et al., 2002; DalCorso et al., 2008) or the NADH dehydrogenase-like complex (NDH; for review, see Battchikova et al., 2011; Ifuku et al., 2011). The functioning of either CEF pathway, which generates a pH gradient ΔpH without any accumulation of NADPH, is thought to achieve the appropriate ATP/NADPH balance required for the biochemical needs of the plant, especially under certain environmental conditions such as low CO₂ (Golding and Johnson, 2003), heat (Clarke and Johnson, 2001), cold (Clarke and Johnson, 2001), drought (Golding and Johnson, 2003; Kohzuma et al., 2009), high light (Munekage et al., 2004), or dark-to-light transitions (Joliot and Joliot, 2005; Fan et al., 2007). CEF-generated ΔpH is also involved in photoprotection owing to the down-regulation of PSII via nonphotochemical quenching (Munekage et al., 2004; Takahashi et al., 2009). Very recently, the role of the PGR5 protein as a regulator of LEF has been established. It has proved to be essential in the protection of PSI from photodamage (Suorsa et al., 2012).The two cyclic pathways are redundant (Munekage et al., 2004), sharing ferredoxin (Fd) as a common stromal electron donor (Yamamoto et al., 2011) and electron carriers from plastoquinone (PQ) to PSI with LEF. Thus, LEF and either of the CEF pathways may be in competition. The molecular events that allow CEF to challenge LEF remain enigmatic, particularly when considering that the conditions that require CEF are also those under which LEF is in excess. Efforts to understand the appropriate functioning of CEF have led to the proposition of several models segregating cyclic and linear pathways at a structural level (for review, see Eberhard et al., 2008; Cardol et al., 2011; Johnson, 2011; Rochaix, 2011). According to the restricted diffusion model, founded on the uneven distribution of the photosynthetic protein complexes in the thylakoids, there is little competition between CEF and LEF, as CEF occurs in stroma lamellae where PSI is concentrated while LEF takes place in the grana stacks. In line with the supercomplex model, whose relevance was demonstrated in the microalga Chlamydomonas reinhardtii, CEF happens within tightly bound supercomplexes containing PSI, with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), cytochrome b₆/f, Fd, Fd NADP reductase (FNR), and the integral membrane protein PGRL1 (Iwai et al., 2010). In higher plants, an association between NDH and PSI subunits suggests the formation of such supercomplexes (Peng et al., 2009). The availability of FNR, found either free in the stroma or bound to the thylakoids (Zhang et al., 2001), has also been proposed to modulate partitioning between LEF and CEF (Joliot and Joliot, 2006; Joliot and Johnson, 2011). In addition, more dynamic models that illustrate competitive processes involved in the distribution of electrons between the cyclic and linear flows have been proposed. The competition between cytochrome b₆/f and FNR for electrons from Fd could regulate the segregation between LEF and CEF (Breyton et al., 2006; Yamamoto et al., 2006; Hald et al., 2008). A few years ago, Joliot and Joliot (2006) suggested that the ATP/ADP ratio was one of the parameters that triggered on the transition between LEF and CEF. It was also established that the redox poise of chloroplast stroma contributed to the regulation of the photosynthetic pathway and played an important role in defining the extent of CEF. Breyton et al. (2006) scrutinized this redox regulation and established that the fraction of PSI complexes engaged in CEF could be modulated by changes in the stromal redox state. Overreduction of the NADPH pool was involved in the repartition between LEF and CEF (Joliot and Joliot, 2006). The NADPH/NADP⁺ ratio was proposed as a regulator of PGR-dependent CEF in vivo (Okegawa et al., 2008).All the published data supporting a role for the redox status in the regulation of CEF urged us to investigate a putative role of thioredoxins (TRX) in the regulation of CEF. TRX are ubiquitous disulfide reductases regulating the redox status of target proteins (for review, see Lemaire et al., 2007; Meyer et al., 2009). In chloroplast, TRX mediate the light regulation of numerous enzymes, among which some belong to the Calvin cycle (for review, see Schürmann and Buchanan, 2008; Montrichard et al., 2009; Lindahl et al., 2011). Global proteomic approaches have revealed that well-known photosynthetic complex subunits may be partners of TRX, such as PsbO in PSII, plastocyanin, Rieske Fe-S protein in cytochrome b₆/f, and PsaK and PsaN in PSI (for review, see Montrichard et al., 2009; Lindahl et al., 2011). Furthermore, regarding the regulation of photosynthesis, TRX have also been involved in state transitions (Rintamäki et al., 2000; Buchanan and Balmer, 2005), and their participation in the control of the redox poise of the electron transport chain has also been suggested (Johnson, 2003).In this work, we have investigated the possible role of TRX in the regulation of CEF. Using Arabidopsis (Arabidopsis thaliana) mutants with altered expression of genes encoding different plastid TRX, we have established in vivo the inhibitor activity of TRXm4 on the NDH-dependent pathway for plastoquinone reduction. This result was confirmed in transplastomic tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. Moreover, in vitro assays performed with isolated chloroplasts indicated that TRXm4 negatively controls the PGR-dependent electron flow as well.     

The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4^Cdt2)–PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2–proteasome pathway to facilitate TLS pathway.     

Diversity of the fsr-gelE region of the Enterococcus faecalis genome but conservation in strains with partial deletions of the fsr operon

Most Enterococcus faecalis isolates carry gelE, but many are gelatinase nonproducers due to the lack of fsrC (EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activates gelE expression. Analysis of 22 accessible E. faecalis genomes revealed the identity of the 53-amino-acid propeptide of fsrD across multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen in fsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surrounding fsrC-EF_1841. However, analysis of five sequenced strains carrying the fsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) in gelE and an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conserved gelE SNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kb fsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown by fsr-gelE mutants. In conclusion, we describe the identity of fsrD despite high plasticity within the fsrC-EF_1841 region and the surrounding sequence. However, strains lacking the fsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and in gelE, suggesting that the deletion may result from horizontal transfer and recombination.